Two general modes of amplitude modulation of the cardiac contraction are a variation (a) in the number of Ca(2+) ions made available to bind to the myofilaments during the transient, increase in cytosolic calcium concentration (Cai) following excitation and (b) in the extent of myofilament displacement or force production in response to a given Cai transient. In order, to determine the extent to which a change in the twitch contraction amplitude in intact cardiac cells or muscle is due to either mechanism contraction needs to be assessed simultaneously with measurements of the Cai transient. A direct assessment of the relationship between Cai and cell length, and thus myofilament length, can be made in the absence of external mechanical loading in individual isolated cardiac cells. We have designed a method to simultaneously measure contraction and Cai, as reported by the fluorescent Ca(2+) probe, indo-1 in individual cardiac cells to examine the relationship between Ca(2+) and contraction. During relaxation of the twitch contraction originating from slack length under a given set of conditions, a unique relation exists between cell length and Ca1 during twitches that vary in their individual amplitudes manifest as a single trajectory in the cell length-Ca1 or indo-fluorescence phase plane diagrams. This Cai-length relationship during electrically stimulated twitches is steeper than that described by peak contraction amplitude versus peak Cai relationship same as that due to increases in Cai and contractions elicited via the abrupt and transient application of caffeine or by 'tetanization" of the cell in the presence of ryanodine. This unique relationship between Ca 2+-dependent fluorescence and length shifts appropriately in response to perturbations that have previously demonstrated to alter the steady-state myofilament Ca(2+) sensitivity in skinned cardiac fibers. Thus, the Cai cell length trajectory during the relaxation phase of contraction appears to define a quasi-equilibrium of cytosolic Ca(2+) and myofilament Ca(2+) binding and may be used to assess the myofilament Ca(2+) response during the twitch contraction in single cardiac myocytes.